JP2021107404A - Compositions and methods for treating lysosomal storage disorders - Google Patents

Abstract

To provide a novel pharmaceutical composition useful for treatment of a lysosomal storage disorder, represented by Tay-Sachs disease and Niemann-Pick disease.SOLUTION: The present disclosure provides a composition for treatment of a lysosomal storage disorder. The composition includes a therapeutically effective amount of an agent that mediates upregulation of Transcription Factor EB. The agent is cinnamic acid or sodium benzoate. The composition may further include a therapeutically effective amount of all-trans retinoic acid or vitamin A.SELECTED DRAWING: Figure 1

Description

Related Applications This application claims priority under US Patent Provisional Application No. 62 / 081,696 filed on November 19, 2014, and the entire disclosure of the above application is incorporated herein by reference.
The present invention relates to compositions and methods for the treatment of lysosomal storage diseases as a whole.

Lysosomes are membrane-bound organelles containing many hydrolases that are highly active in an acidic environment (1-3). Lysosomes were classically identified as organelles that manage waste products,
It has been shown to be involved in major intracellular processes such as degradation, programmed cell death, and trophic response during development (2,4-8). The diverse roles of lysosomes and their responses to different stimuli suggest a coordinated regulation of lysosomal gene expression (9,10). Recent studies have provided information on the regulation of lysosomal genes (11,12), but pattern-finding analysis of lysosomal genes has provided information on the bHLH (basic helix-loop-helix) factor small eyeball transcription factor. Coordinated Lysosomal Expression, a potential binding site for the transcription factor EB (TFEB), a member of the E (MiT / TFE) subfamily.
and Regulation (CLEAR)) The existence of the element has been clarified. Studies have reported a possible link between TFEB and lysosomal biosynthesis (9,10,12).

The regulation of Tfeb is complex and appears to be cell type and stimulus dependent. In differentiated osteoclasts, the RANKL-dependent signaling pathway induces TFEB activation-induced lysosomal biosynthesis (13). Starvation or stress conditions can also activate TFEB, which is normally kept inactive by mTORC1 (14,15). In one study, starvation-induced TFEB activity played an important role in lipid metabolism, and activated TFEB
Has also been shown to be able to self-regulate its own gene expression (16).

Lysosomal storage disease (LSD) is a group of approximately 50 rare hereditary metabolic disorders resulting from defective lysosomal function. Symptoms of LSD vary depending on the particular disease and other variable factors, such as age of onset, and can be mild to severe. Symptomatology may include stunted growth, movement disorders, seizures, dementia, deafness and / or blindness. Some people with LSD have liver enlargement (hepatomegaly), spleen enlargement (splenomegaly), lung and heart disease, and abnormally growing bones.

Overview of Preferred Embodiments One aspect of the invention provides a method of treating LSD. The method may include administering to a subject in need of treatment a composition containing a therapeutically effective amount of an agent that mediates upregulation of the transcription factor EB (TFEB).

In one embodiment, the agent is a statin. For example, statins include atrovastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin,
Or it can be simvastatin. The agent can also be, for example, a lipid lowering agent, such as a fibrate. In some embodiments, the fibrate is gemfibrozil or fenofibrate. In other embodiments, the agents are analgesics, antipyretics, aspirin, cinnamon (ci).
nnamon) Metabolite, cinnamic acid, sodium phenylbutyrate or sodium benzoate. In yet another embodiment, the composition may contain a combination of at least two of the above agents.

The composition may also contain all-trans retinoic acid or vitamin A. for example,
The composition is statin or fibrate and all-trans retinoic acid or vitamin A.
Can be contained. This combination of the drug with all-trans retinoic acid or vitamin A can provide greater therapeutic effect in the subject than administration of all-trans retinoic acid, vitamin A or fibrates alone. The combination can be a synergistic combination. TFEB can also be upregulated by elevated transcription factor EB mRNA levels, elevated transcription factor EB protein levels, or activation of the PPARa-RXRa heterodimer.

LSD includes neurodegenerative diseases such as Parkinson's plus diseases such as neuroseloid lipofustinosis, Alzheimer's disease, Huntington's disease, muscular atrophic lateral sclerosis (ALS), multiple system atrophy (MSA). It can be Parkinson's disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) or Lewy body dementias (DLB). In another embodiment, LSD is a disorder of the autophagy pathway and the agent increases lysosomal biosynthesis.

In other embodiments, the LSD is Tay-Sachs disease, Fabry disease, Niemann-Pick disease, Gaucher disease, Hunter syndrome, α-mannose disease, Aspatyl glucosamine urine disease (Aspa).
rtylglucosaminuria), Cholesteryl ester storage di
sease), chronic hexosaminidase A deficiency, cystinosis, Danon disease, Farber's disease, fucosidosis, galactosialidosis, or Batten disease including late-onset childhood Batten disease and juvenile Batten disease.

Another aspect of the invention provides a method of treating LSD, comprising administering to a subject in need of treatment a composition containing a therapeutically effective amount of an agent that mediates upregulation of the Tfeb gene. .. Yet another aspect provides a method of treating LSD, comprising administering to a subject in need of treatment a composition containing a therapeutically effective amount of an agent that restores TFEB activity.

We show that gemfibrozil and retinoic acid upregulate TFEB mRNA and protein levels in brain cells. (A, B) Mouse primary stellate cells were treated with different concentrations of gemfibrozil and all-trans retinoic acid (ATRA) under serum-free DMEM / F-12 medium for 12 hours, followed by tfeb mRNA levels by qRT-PCR. (A), TFEB protein levels (B) were monitored by immunoblot. (C) Densitometric analysis of immunoblots for TFEB (compared to β-actin). (D, E) Mouse primary stellate cells were treated with a combination of gemfibrozil (10 μM) and ATRA (0.2 μM) for 4, 6, 12 and 24 hours under similar culture conditions before TFEB mRNA levels by qRT-PCR. (D), protein level (E) was monitored by immunoblotting. (F) Densitometry analysis of immunoblots for TFEB. All results show the mean ± SEM of at least 3 independent experimental sets. (G, I) Mouse primary astrocytes (G) and mouse primary neurons (I) were treated with a combination of gemfibrozil and retinoic acid under serum-free conditions for 24 hours, respectively, TFEB (red) -GFAP (green) and Double labeled with TFEB (red) -Map2 (green). DAPI was used for nuclear staining. Scale bar = 20 μm (G), scale bar = 5 μm (G) for high-magnification images; scale bar = 50 μm (I), scale bar = 10 μm (I) for high-magnification images. (H, J) Quantification of TFEB immunoreactivity (TFEB IR) in whole cells and nuclei of mouse primary astrocytes (H) and mouse primary neurons (J) as a magnification to control is shown. From three independent experimental sets, ImageJ is used to quantify at least 25 separate images for each condition. t: p <0.05 (for untreated controls). The involvement of PPARα and RXRα in fibrate-mediated upregulation of TFEB mRNA and protein is shown. (A, B): Gemfibrozil (10 μM) and retinoic acid (0.2 μM) in serum-free DMEM / F12 for 24 hours in mouse primary stellate cells isolated from PPARα-/-and PPARβ-/-and wild-type mice. After treatment with the combination of), Tfeb mRNA expression was monitored by real-time PCR (A) and TFEB protein levels were monitored by immunoblot (B). (C): Shows densitometric analysis of TFEB levels (compared to β-actin) in PPARα-/-and PPARβ-/-and wild-type astrocytes. a: p <0.05 (for WT control); b: p <0.05 (for PPARβ-/-control); ns: not significant for PPARα-/-control. (D): Mouse primary astrocytes isolated from WT mice were pretreated with GW9662 for 30 minutes, then treated with gemfibrozil and retinoic acid under similar culture conditions, followed by immunoblotting to determine the level of TFEB protein expression. Monitored. (E): Shows densitometric analysis of immunoblots of TFEB (compared to β-actin). t: p <0.05 (relative to control); ns: not significant for control. (F): Mouse primary astrocytes isolated from PPARα-/-and PPARβ-/-and WT mice were treated in serum-free DMEM / F12 for 24 hours with 10 μM gemfibrozil and 0.2 μM retinoic acid. Double labeled for TFEB (red) and GFAP (green). DAPI was used to stain the nuclei. UN: No processing. Scale bar = 20 μm. (G): Shows the quantification of TFEB immune reactivity (TFEB IR) in mouse primary astrocytes calculated as a magnification to control. From three independent experimental sets, at least 25 separate images per condition were quantified using ImageJ. a: p <0.05 (for WT control); b: p <0.05 (for PPARβ-/-control); ns: not significant for PPARα-/-control. (H, I, J): Mouse primary stellate cells transfected or untransfected with scrambled siRNA (1.0 μg) or RXRα siRNA (1.0 μg) for 36 hours, followed by serum-free DMEM / F12 medium. After treatment with a combination of RA (0.2 μM) and gemfibrodil (10 μM) for 24 hours in, RT-PCR (H) for RXRα, quantitative real-time PCR (J) for TFEB to check the level of gene silencing. ), And immunoblot (J) for TFEB. (K): Shows densitometric analysis of immunoblots for TFEB (compared to β-actin). *: P <0.05 (for untransfected controls); **: p <0.05 (for scrambled siRNA-transfected controls); ns: Not significant for RXRα siRNA-transfected controls. All results represent the mean ± SEM of at least 3 independent experimental sets. It is shown that PPARα transcriptionally regulates TFEB expression under treatment conditions. (A): Shows a map of wild-type and mutant PPRE sites of the TFEB-luciferase promoter construct. (B): BV2 cells were transfected with pTFEB (WT) -Luc for 24 hours, then treated with different concentrations of gemfibrozil and retinoic acid alone or in combination and subjected to a luciferase assay. *: P <0.05 (for untreated control). (C): BV2 cells were transfected with pTFEB (WT) -Luc for 24 hours, pretreated with PPARα-, PPARβ-, PPARγ-antagonists, followed by treatment with gemfibrozil and retinoic acid for luciferase assay. did. *: P <0.05 (for untreated control). #: p <0.05 (for processing). (D): Mouse primary stellate cells isolated from PPARα-/-(center) and PPARβ-/-(right) and wild-type (left) mice were transfected with pTFEB (WT) -Luc for 24 hours and then transfected. It was treated with gemfibrozil and retinoic acid and subjected to a luciferase assay. *: P <0.05 (for untreated WT control). #: Not significant for untreated PPARα-/-control. t: p <0.05 (for untreated PPARβ-/-control). (E, F): BV2 cells (E) and mouse primary stellate cells (F) were transfected with pTFEB (WT) -Luc and pTFEB (Mu) -Luc for 24 hours and then treated with gemfibrozil and retinoic acid. , Was subjected to a luciferase assay. *: P <0.05 (for untreated pTFEB (WT) -Luc transfect control). ns: Not significant for untreated pTFEB (Mu) -Luc transfected controls. All results are mean ± SEM of at least 6 independent experimental sets. Transcriptional activation of TFEB by the PPARα-RXRα-PGC1α complex is shown. (A): Shows the map of PPRE on the TFEB promoter with the core sequence and the amplicon length of ChIP. (B, C): Mouse stellate cells treated with a combination of gemfibrozil (10 μM) and RA (0.2 μM) for 30, 60, 120, and 240 minutes and PPARα on the PPRE binding site of the Tfeb promoter (leftmost). ), RXRα (second from left), PGC1α (second from right) and RNA polymerase (far right) were monitored by RT-PCR (B) and qRT-PCR (C) after ChIP. .. Normal IgG was used as a control. *: P <0.05 (for untreated control). All results represent the mean ± SEM of at least 3 independent experimental sets. (D): Shows a schematic diagram of induction of lysosomal biosynthesis by activating a peroxisome proliferating agent. We show that PPARα-dependent upregulation of TFEB induces lysosomal biosynthesis. (A, B): Mouse primary stellate cells isolated from PPARα-/-and PPARβ-/-and wild-type mice in serum-free DMEM / F12 for 24 hours with gemfibrozil (10 μM) and retinoic acid (0.2 μM). ), Then the mRNA expression of lysosomal genes (Lamp2 (left), Limp2 (center), Npc1 (right)) was monitored by real-time PCR (A) and the protein level of LAMP2 was monitored by immunoblot (B). .. (C): Shows densitometric analysis of LAMP2 levels (compared to β-actin) in PPARα-/-and PPARβ-/-and wild-type astrocytes. a: p <0.05 (for WT control); b: p <0.05 (for PPARβ-/-control); ns: not significant for PPARα-/-control. All results represent the mean ± SEM of at least 3 independent experimental sets. (D): Mouse primary astrocytes isolated from PPARα-/-and PPARβ-/-and WT mice were treated in serum-free DMEM / F12 for 24 hours with 10 μM gemfibrozil and 0.2 μM retinoic acid. Double labeled for LAMP2 (red) and GFAP (green). DAPI was used to stain the nuclei. (E): Quantification of LAMP2 immune reactivity (Lamp2 IR) of mouse primary astrocytes calculated as a magnification with respect to the control. a: p <0.05 (for WT control); b: p <0.05 (for PPARβ-/-control); ns: not significant for PPARα-/-control. (F): Primary mouse neurons isolated from WT mice were treated in serum-free DMEM / F12 for 24 hours with 10 μM gemfibrozil and 0.2 μM retinoic acid and doubled for LAMP2 (red) and Map2 (green). Signed. DAPI was used to stain the nuclei. (G): Shows the quantification of LAMP2 immune responsiveness (Lamp2 IR) of primary mouse neurons calculated as a magnification relative to the control. *: P <0.05 (for untreated control). (H): Mouse primary astrocytes isolated from PPARα-/-and PPARβ-/-and WT mice were treated in serum-free DMEM / F12 for 24 hours with 10 μM gemfibrozil and 0.2 μM retinoic acid. Double labeled for lithotrackers red (red) and GFAP (green). (I): Quantification of LAMP2 immune reactivity (Lamp2 IR) of mouse primary astrocytes calculated as a magnification with respect to the control. a: p <0.05 (for WT control); b: p <0.05 (for PPARβ-/-control); ns: not significant for PPARα-/-control. UN: No processing. Scale bar = 20 μm (for D, E & F), scale bar for high-magnification images = 10 μm (for E). At least 25 images per condition from three different experimental sets were analyzed using ImageJ for all image quantitative data. It is shown that oral administration of gemfibrozil upregulates TFEB in the cortex of WT and PPARβ-/-mice in vivo, but not in PPARα-/-mice. (A, D, G): WT, PPARα-/-and PPARβ-/-mice (n = 6 in each group) were forcibly fed with 7.5 mg / kg body weight / day of gemfibrozil and 0.1 mg / kg body weight of all-trans. Treated with type retinoic acid (dissolved in 0.1% methylcellulose) or vehicle weight (0.1% methylcellulose). After 60 days of treatment, mice were sacrificed and cortical sections were double labeled for TFEB (red) and NeuN (green). DAPI was used to visualize the nucleus. (C, F, I): High-magnification images showing the localization of TFEB and NeuN in cortical neurons of treated group mice (WT, PPARα-/-and PPARβ-/-). (B, E, H): Shows quantification of TFEB immunoreactivity (TFEB IR) in untreated and treated samples from each group (WT, PPARα-/-and PPARβ-/-), expressed as% area. a: p <0.05 (for WT control); b: p <0.05 (for PPARβ-/-control); ns: not significant for PPARα-/-control. At least 12 sections (2 sections per animal) from each group were quantified using ImageJ. Scale bar = 50 μm and 10 μm (for high magnification images). It is shown that oral administration of gemfibrozil upregulates LAMP2 in the cortex of WT and PPARβ-/-mice in vivo, but not in PPARα-/-mice. (A, D, G): WT, PPARα-/-and PPARβ-/-mice (n = 6 in each group) were forcibly fed with 7.5 mg / kg body weight / day of gemfibrozil and 0.1 mg / kg body weight of all-trans. Treated with type retinoic acid (dissolved in 0.1% methylcellulose) or vehicle weight (0.1% methylcellulose). After 60 days of treatment, mice were sacrificed and cortical sections were double labeled for LAMP2 (red) and NeuN (green). DAPI was used to visualize the nucleus. (C, F, I): High-magnification images showing the localization of LAMP2 and NeuN in cortical neurons of mice (WT, PPARα-/-and PPARβ-/-) in the treatment group. (B, E, I): Shows the quantification of LAMP2 immunoreactivity (LAMP2 IR) in untreated and treated samples from each group (WT, PPARα-/-and PPARβ-/-), expressed as% area. a: p <0.05 (for WT control); b: p <0.05 (for PPARβ-/-control); ns: not significant for PPARα-/-control. At least 12 sections (2 sections per animal) from each group were quantified using ImageJ. Scale bar = 50 μm and 10 μm (for high magnification images). We show that upregulation of TFEB induces lysosomal biosynthesis in both normal and LINCL patient fibroblasts. Fibroblasts of healthy individuals (WT # 1-3), LINCL patients (NCL # 1-5) and LINCL carriers (NCL / C) were placed in low serum (2%) DMEM medium for 24 hours with gemfibrozil (Gemfibrozil). After treatment with 10 μM) and retinoic acid (0.2 μM), it was stained with lithotracker red (red). A brightfield microscope was used to detect cell morphology. Scale bar = 20 μm. The corresponding boxplot shows multiple changes in the lithotracker positive signal of the treatment group relative to the control in each cell type. *: P <0.05 (for untreated control). ROI-The white dotted line represents the area of the cell. Magnification changes were calculated as Lyso T-positive signals per unit area per cell in the treated group relative to the control. At least 25 individual images per cell type condition were quantified using ImageJ. (A)-(D) are cholesterol-lowering agents (simvasstatin and pravastatin), aspirin (analgesics and antipyretics), cinnamic acid (metabolite of cinnamic acid), and agents for urea cycle disorders (sodium phenylbutyrate and benzoate). The up-regulation of TFEB mRNA expression in mouse stellate cells by sodium) is shown. It shows an increase in lysosomal biosynthesis by aspirin in primary mouse astrocytes. Cells were treated with different concentrations of aspirin under serum-free conditions for 24 hours before lithotracker staining. The results represent three independent experiments. (A)-(D) show upregulation of LAMP2 expression by aspirin in primary mouse astrocytes. 11 (A): Cells were treated with 5 μM aspirin for different times under serum-free conditions and then LAMP2 mRNA expression was monitored by real-time PCR. The result is the average of 3 different experiments + SD. a: p <0.05 (for control); b: p <0.001 (for control). 11 (B): Cells were treated with different concentrations of aspirin for 24 hours under serum-free conditions before Western blotting for LAMP2. 11 (C): Cells were treated with 5 μM aspirin for different times under serum-free conditions before Western blotting for LAMP2. 11 (D): After 24 hours of aspirin treatment, cells were double labeled for LAMP2 and GFAP. The results represent three independent experiments. (A)-(C) show an increase in TPP1 by aspirin in primary mouse astrocytes. 12 (A): Cells were treated with different concentrations of aspirin for 24 hours under serum-free conditions before Western blotting for TPP1. 12 (B): Cells were treated with 5 μM aspirin for different times under serum-free conditions before Western blotting for TPP1. Actin was run as a housekeeping molecule. 12 (C): Cells were treated with different concentrations of aspirin for 24 hours under serum-free conditions, followed by cell extracts containing 5 μg of total protein and Ala-Ala-Phe7-amide-4-methylcoumarin as substrate. TPP1 activity assay was performed using. The results represent three independent experiments. (A)-(C) show upregulation of TFEB by aspirin in primary mouse astrocytes. 13 (A): Cells were treated with different concentrations of aspirin for 12 hours under serum-free conditions and then Western blots were performed on TFEB. Actin was run as a housekeeping molecule. 13 (B): Cells were treated with 5 μM aspirin for 12 hours under serum-free conditions and then double labeled with GFAP and TFEB. These results are the average of two independent experiments. 13 (C): Cells were transfected with the p (WT) Tfeb-Luc plasmid and 24 hours after transfection, the cells were stimulated with different doses of aspirin. After 4 hours, firefly luciferase activity in whole cell extract was measured. The result is the average of 3 different experiments + SD. a: p <0.001 (relative to control). (A)-(B) show activation of PPARα by aspirin in primary mouse astrocytes. 14 (A): To monitor the DNA binding activity of PPARα using the PPARα-binding site of the Tfeb promoter as a probe to isolate the nuclear extract after treating the cells with 5 μM aspirin for different hours (minutes). Electrophoretic mobility shift assay was performed. 14 (B): Stellate cells isolated from wild-type, PPARα (-/-) and PPARβ (-/-) mice were transfected with the PPAR luciferase reporter (PPRE-x3-TK-luc) plasmid and transfected. Twenty-four hours after transfection, cells were stimulated with different doses of aspirin. After 4 hours, firefly luciferase activity in whole cell extract was measured. The result is the average of 3 different experiments + SD. a: p <0.001 (relative to control). (A)-(C) show that aspirin increases the level of TFEB in astrocytes via PPARα. Astrocytes isolated from WT 15 (A), PPARα (-/-) 15 (B) and PPARβ (-/-) 15 (C) mice were treated with 5 μM aspirin for 12 hours under serum-free conditions. After that, TFEB and GFAP were double-labeled. The results represent three independent experiments. (A)-(B) show that aspirin increases the level of LAMP2 in astrocytes via PPARα. Astrocytes isolated from WT, PPARα (-/-) and PPARβ (-/-) mice were treated with different concentrations of aspirin for 24 hours under serum-free conditions and then Western blot analyzed for LAMP2 (16). (A)). Actin was run as a housekeeping molecule. Bands were scanned and compared to controls (16 (B)). The result is the average of 3 different experiments + SD. a: p <0.05 (for control); b: p <0.001 (for control). ns: Not significant. (A)-(C) show that aspirin increases lysosomal biosynthesis in astrocytes via PPARα. Astrocytes isolated from WT 17 (A), PPARα (-/-) 17 (B) and PPARβ (-/-) 17 (C) mice were treated with 5 μM aspirin for 24 hours under serum-free conditions. After that, it was stained with lithotracker. The results represent three independent experiments.

Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In the event of conflict, the description, including the definitions herein, will prevail. Preferred methods and substances are described below, but methods and substances similar or equivalent to those described herein can be used in the practice or testing of the present invention.

The use of the terms "a" and "an" and "the" and similar terms in the context in which the invention is described (particularly in the context of the claims below) is used unless otherwise indicated herein or by context. Unless there is a clear contradiction, it should be construed to include both singular and plural. The description of a numerical range herein is intended to serve solely as an abbreviation for individual references to distinct numerical values contained within that range, unless otherwise indicated herein. , Each separate number is incorporated herein by reference as individually described herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or where there is no apparent contradiction in the context. The use of any and all examples, or exemplary terms (such as "etc.", "eg", etc.) provided herein is merely intended to better illustrate the invention, and others. Does not impose any restrictions on the scope of the invention unless otherwise claimed. Nothing in the specification should be construed as indicating an element not stated in the claims as essential to the practice of the present invention.

As used herein, the term "subject" refers to a human or veterinary subject. As used herein, the term "therapeutic effect" induces or alleviates a subject's disease, eg, pathological symptoms associated with LSD, improvement in disease progression or physiological status, or resistance to disease. Or it means the effect that causes. The term "therapeutically effective amount" used in connection with a drug means the amount of drug that has a therapeutic effect on a subject.

The terms "synergistic", "synergistic" or "synergistic" mean greater than the additive effect expected in a combination. The synergistic effect is that the active ingredients are (1) formulated together in a combined unit-dose formulation and administered or delivered simultaneously, or (2) delivered alternately or in parallel as separate formulations. (3) It can be achieved if it is due to other regimens.

Compositions and Methods for the Treatment of Lysosomal Storage Disease For the purpose of facilitating an understanding of the principles of the present invention, embodiments are referred to below, some of which are shown in the drawings and specific terminology used to describe them. do. However, it will be understood that these are not intended to limit the scope of the invention. It is envisioned that any modification and further modification in the described embodiments, and any further application of the principles of the invention described herein, will be made normally by an expert in the field to which the invention relates. NS.

One aspect of the invention relates to a method of treating lysosomal storage disease (LSD). LSD includes, for example, Tay-Sachs disease, Fabry disease, Niemann-Pick disease, Gaucher disease, Hunter syndrome, α.
Mannose disease, aspartyl glucosamineuria, cholesterol ester accumulation disease, chronic hexosaminidase A deficiency, cystinosis, Danon disease, Farber's disease, fucosidosis, galactosialidosis, or late-onset childhood Batten disease and juvenile Batten It can be Batten's disease, including the disease. LSD is also a neurodegenerative disease involved in the autophagy-lysosomal pathway,
Parkinson's disease, including Parkinson's-like diseases such as neuroceroid lipofustinosis, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis (ALS), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) , Corticobasal degeneration (CBD) or Lewy body dementias (DLB)
Can be. Neurodegenerative diseases can be characterized by incomplete autophagy. Such diseases include Alzheimer's disease, Parkinson's disease, and Huntington's disease.

One embodiment comprises administering to a subject suffering from LSD an agent that upregulates or increases expression from the Tfeb gene. Upregulation is Tfeb's mRN
May include raising the A level. The methods of the invention also include administering to a subject suffering from LSD an agent that upregulates TFEB or restores TFEB activity. Upregulation can include raising TFEB mRNA levels, raising TFEB protein levels, or raising TFEB activity. Activation of the PPARα / RXRα heterodimer results in upregulation of TFEB. We also surprisingly upregulate TFEB through the activity or involvement of PPARα, but PPARβ or PPARγ.
Showd that it was not up-regulated.

The agent can be a lipid lowering agent such as fibrates. The fibrate can be gemfibrozil, fenofibrate, or clofibrate. The agent can be all-trans retinoic acid or vitamin A. Surprisingly and unexpectedly, administration of fibrates to subjects in combination with all-trans retinoic acid or vitamin A produces TFEB rather than administration of fibrates or all-trans retinoic acid or vitamin A alone. Can be more up-regulated. When fibrates and all-trans retinoic acid or vitamin A are co-administered to a subject, they can jointly promote upregulation of TFEB and synergistically upregulate TFEB. The dose of fibrate that may be required to achieve the same degree of TFEB upregulation that occurs only when higher doses of fibrate are given to the subject is higher in the presence of all-trans retinoic acid or vitamin A. low. The combination of fibrates and all-trans retinoic acid or vitamin A can be a synergistic combination.

In another embodiment, the lipid lowering agent is a statin. For example, the statin can be atrovastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, or a combination of at least two of these agents. 1
More than a species of statin can be used alone or in combination with fibrates and all-trans retinoic acid or vitamin A. In yet another embodiment, the agent can be an analgesic or antipyretic, such as aspirin; phenylbutyrate; sodium benzoate; or a cinnamon metabolite, such as cinnamic acid. Such agents can also be used in combination with all-trans retinoic acid or vitamin A and administered together to the subject to jointly promote upregulation of TFEB to synergistically increase TFEB. Can be regulated.

Lipid lowering agents can be agents that reduce the levels of triglycerides circulating in a subject's blood. In addition, lipid-lowering agents can be agents that reduce the risk of dyslipidemia. Fibrates can mediate upregulation of TFEB via PPARα, but PPARβ and PPAR
It does not mediate via γ. During TFEB upregulation, PPARα forms a heterodimer with RXR-α, and the RXRα / PPAR-a heterodimer is recruited to the promoter of the Tfeb gene via the RXR binding site.

Upregulation of TFEB can also be mediated by all-trans retinoic acid. All-trans retinoic acid is also known as ATRA, retinoic acid, tretinoin, and vitamin A acid. All-trans retinoic acid can mediate upregulation of TFEB via the retinoid X receptor-α (RXR-α). During TFEB upregulation, RXR-α forms a heterodimer with the peroxisome proliferator-activated receptor-a (PPAR-α), and the RXR-α / PPAR-α heterodimer is the RXR binding site. It is recruited to the promoter of the Tfeb gene via.

Compositions that mediate upregulation of TFEB may contain a combination of a drug, such as a lipid lowering agent, with all-trans retinoic acid or vitamin A. Such a combination may co-mediate or increase the upregulation of TFEB as compared to administration of the drug or all-trans retinoic acid or vitamin A alone. This combination is compared to administration of lipid-lowering agents or all-trans retinoic acid or vitamin A alone, TFEB
Upregulation can be increased jointly by about 2 times, about 3 times, about 4 times, about 5 times, or about 10 times. In particular, this combination can jointly increase TFEB upregulation by about 3-fold compared to administration of lipid-lowering agents or all-trans retinoic acid or vitamin A alone.

Another aspect of the invention provides a pharmaceutical composition containing at least one of the above agents.
The pharmaceutical composition may also contain all-trans retinoic acid or vitamin A. For example, the pharmaceutical composition may contain gemfibrozil, or a combination of gemfibrozil and all-trans retinoic acid or vitamin A, or aspirin and all-trans retinoic acid or vitamin A.

Pharmaceutical compositions include, for example, tablets, rounds, sugar-coated tablets, hardgel and softgel capsules, granules, pellets, aqueous, lipid, oily or other solutions, emulsions such as oil-in-water emulsions, liposomes, aqueous or oily suspensions. It can be in the form of liquids, syrups, alixiers, solid emulsions, solid dispersions or dispersible powders. In pharmaceutical compositions for oral administration, the agents are commonly known and used auxiliaries and excipients such as arabic gum, talc, starch, sugars (eg mannitose, methylcellulose, lactose). , Gelatin, surfactant, magnesium stearate, aqueous or non-aqueous solvent, paraffin derivative, cross-linking agent, dispersant, emulsifier, lubricant, preservative, flavoring agent (eg ether oil), dissolution accelerator (eg benzyl benzoate) Or it can be mixed with a benzyl alcohol) or a bioavailability accelerator (eg GELUCIRE). In pharmaceutical compositions, the drug may also be dispersed in fine particles, eg, nanoparticulate compositions.

For parenteral administration, the drug, or pharmaceutical composition of the drug, is in a physiologically acceptable diluent such as water, buffer, oil with or without solubilizer, surfactant, dispersant or emulsifier. Can be dissolved or suspended in. As the oil, for example, olive oil, peanut oil, cottonseed oil, soybean oil, castor oil and sesame oil can be used without limitation. More generally, for parenteral administration, the drug, or pharmaceutical composition of the drug, can be in the form of aqueous, lipid, oily or other types of solutions or suspensions, or liposomes or nanosuspends. It can also be administered in the form of a turbid solution.

Method of Administration The above agents, or pharmaceutical compositions containing these agents, can be administered by any method that allows a therapeutically effective amount of the agent to be delivered to the subject. Methods of administration include, but are not limited to, oral, topical, transdermal and parenteral routes, as well as direct tissue infusion and catheter delivery. Parenteral routes include, but are not limited to, subcutaneous, intradermal, intra-articular, intravenous, intra-abdominal, and intramuscular routes. In one embodiment, the route of administration is by topical or transdermal administration, eg, by lotion, cream, patch, injection, implant device, graft or other controlled release carrier. Routes of administration include any route that delivers the composition directly to the systemic circulation (eg, by injection), including any parenteral route.

One embodiment of the method of the invention comprises at least one agent, eg gemfibrozil or gemfibrozil and ATRA, at a dose, concentration and time sufficient to prevent the development of LSD or reduce the degree of LSD. Including administering the combination. Certain embodiments are at least 1 at doses of about 0.1 μg to about 100 mg / kg body weight of the subject, about 0.1 μg to about 10 mg / kg body weight of the subject, and about 0.1 μg to about 1 mg / kg body weight of the subject. Includes systemic administration of the seed drug. In practicing this method, the drug, or therapeutic composition containing the drug, can be administered in a single daily dose or in multiple doses per day. This method of treatment may require long-term administration. The amount or total dose administered will be determined by the physician and will depend on factors such as the patient's weight, the patient's age and general condition, and the patient's resistance to the compound.

Embodiments of the present invention will be further described in the following examples, but these do not limit the scope of the invention described in the claims.

[Example 1-Material and method]
Reagents: DMEM / F-12 50/50 1x, Hanks solution (HBSS) and 0.05% trypsin Mediatech (W)
Purchased from ashington, DC). Fetal bovine serum (FBS) is Atlas Biologicals (Fort Collins)
Obtained from s, CO). Antibiotics, antifungal agents, gemfibrozil and all-trans retinoic acid (ATRA) were obtained from Sigma-Aldrich (St. Louis, MO).

Isolation of mouse primary astrocytes: Astrocytes were isolated from mixed glial cell cultures as described (24, 25) according to Giulian and Baker's method (26). Briefly,
On day 9, the mixed glial cell culture was washed 3 times in Dulbecco's modified Eagle's medium / F-12 and shaken in a rotary shaker at 37 ° C. for 2 hours at 240 rpm to remove microglia. Two days later, shaking was repeated for 24 hours to remove oligodendrocytes, ensuring complete removal of all astrocytes. Adhered cells were sprinkled on new plates for further testing.

Isolation of primary mouse neurons: Neurons of mouse fetal (E18-E16) are described above (27).
) Was modified to prepare. The entire brain was removed, cells were washed by centrifugation at 1200 rpm for 10 minutes three times, pellets were dissociated, and 8-wells were pretreated with poly-D-lysine (Sigma, St. Louis, MO) for more than 2 hours. Cells were seeded on chamber slides with 10% confluence. After 4 minutes, the suspension of non-adherent cells was aspirated and 500 ml complete Neurobasal medium (Invitr) supplemented with 2% B27.
ogen) was added to each well. Cells were incubated for 4 days prior to the experiment. Double-labeled immunofluorescence of β-tubulin and GFAP or CD11b revealed that the neurons were ultra-pure 98% (data not shown). Neurobasa with 2% B27 and no antioxidants
Cells were stimulated with gemfibrozil for 24 hours in medium (Invitrogen) for methanol fixation and immunostaining.

Semi-quantitative reverse transcriptase conjugated polymerase chain reaction (RT-PCR): From mouse primary stellate cells and human primary stellate cells, using the RNA-Easy Qiagen (Valencia, CA) kit and following the manufacturer's protocol. RNA was isolated. Semi-quantitative RT-PCR is an oligo (d) as a primer.
T) Moloney murine leukemia virus reverse transcriptase (MMLV-) in 12-18 and 20 μl reaction mixture
RT, Invitrogen) was used as described above (28). Obtained cDNA
The following primers for Promega Master Mix (Madison, WI) and mouse genes (I)
Properly amplified using nvitrogen):

Mouse Tfeb: Sense, 5'-AAC AAA GGC ACC ATC CTC AA-3'(SEQ ID NO: 1); Antisense, 5'-CAG CTC GGC CAT ATT CAC AC-3'(SEQ ID NO: 2); Mouse Lamp2: Sense ,Five'
-GGT GCT GGT CTT TCA GGC TTG ATT-3'(SEQ ID NO: 3); Antisense, 5'-ACC ACC CA
A TCT AAG AGC AGG ACT-3'(SEQ ID NO: 4); Mouse Limp2: Sense, 5'-TGT TGA AAC GG
G AGA CAT CA-3'(SEQ ID NO: 5); Antisense, 5'-TGG TGA CAA CCA AAG TCG TG-3'
(SEQ ID NO: 6); Mouse Npc1: Sense, 5'-GGG ATG CCC GTG CCT GCA AT-3'(SEQ ID NO: 7); Antisense, 5'-CTG GCA GCT ACA TGG CCC CG-3'(SEQ ID NO: 8) ); Mouse Gapd
h: Sense, 5'-GCA CAG TCA AGG CCG AGA AT-3'(SEQ ID NO: 9); Antisense, 5'-G
CC TTC TCC ATG GTG GTG AA-3'(SEQ ID NO: 10).

The amplified product was electrophoresed on a 2% agarose (Invitrogen) gel and visualized by ethidium bromide (Invitrogen) staining. Using glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA as a loading control, it was confirmed that the same amount of cDNA was synthesized from each sample.

Quantitative real-time PCR: mRNA quantification, ABI Prism 7700 Sequence Detection System (Applied B)
SYBR Select master mix (Applied Biosystems) using iosystems, Foster City, CA)
) Was used. Normalize the mRNA expression of the targeting gene to the level of Gapdh mRNA and ABI Se
The data was processed by the quence Detection System 1.6 software.

Immunostaining of cells: Immunocytochemistry was performed as previously reported (29). Briefly, 8-well chamber slides containing primary mouse astrocytes, mouse neurons were cultured to 70-80% confluence and cold methanol (Fisher Scientific, Wal).
It was fixed overnight with tham, MA) and washed twice in a short time with filtered PBS. Sample, Twee
2% BSA (Fisher Scientific) in PBS containing n20 (Sigma) and Triton X-100 (Sigma)
) For 30 minutes and the following anti-mouse primary antibodies: TFEB (1: 1000; Abcam), GFAP, (1: 100)
0; DAKO), LAMP2 (1: 500, Abcam), NeuN (1: 500, Millipore), and MAP2 (1: 200, Millip)
Incubated in PBS containing ore) for 2 hours under shaking conditions at room temperature. 1 in filtered PBS
After washing 4 times for 5 minutes, slide the slides to Cy2 or Cy5-labeled secondary antibody (all 1: 200; Jackson).
Incubated with ImmunoResearch, West Grove, PA) for an additional hour under similar shaking conditions. After four 15-minute washes with filtered PBS, cells were incubated with 4', 6-diamidino-2-phenylindole (DAPI, 1: 10,000; Sigma) for 4-5 minutes. Et sample
It was streamed in an OH and xylene (Fisher) gradient, mounted and observed under an Olympus BX41 fluorescence microscope.

Immunostaining of tissue sections: Mice were sacrificed 60 days after treatment and their brains were fixed, embedded and treated. Sections were made from different brain regions and anti-mouse TFEB (1: 500), anti-mouse LAMP2 (1: 200) and anti-mouse NeuN (1: 500) were used for immunofluorescence staining on fresh frozen sections. .. A sample was placed and observed under an Olympus BX41 fluorescence microscope (30).

LysoTracker staining: Fibroblasts cultured to 70-80% confluence were subjected to various stimuli in serum-reduced (2%) DMEM medium, followed by 75 nM LysoTracker Red DND99. Incubated with (Invitrogen) for 45 minutes.
The cells were then completely washed with filtered PBS, placed on a glass slide and observed under a BX41 fluorescence microscope.

Immune blotting: Western blotting was performed by modifying the above description (31, 32). Briefly, cells are scraped in 1 × RIPA buffer, Br
Proteins were measured using adford reagents, sodium dodecyl sulfate (SDS) buffer was added and electrophoresed on NuPAGE® Novex® 4-12% Bis-Tris gel (Invitrogen) and Thermo. -Proteins were transferred onto a nitrocellulose membrane (Bio-Rad) using Pierce Fast Semi-Dry Blotter.

Membranes were then washed in TBS plus Tween20 (TBST) for 15 minutes and blocked in TBST containing BSA for 1 hour. Next, the following primary antibodies: TFEB (1: 1000, Abcam), LAMP2 (1: 500, Abcam)
Membranes were incubated overnight at 4 ° C. under shaking conditions with and β-actin (1: 800, Abcam, Cambridge, MA). The next day, the membrane was washed in TBST for 1 hour and the secondary antibody against the primary antibody (
All were incubated with Jackson ImmunoResearch for 1 hour at room temperature, washed for an additional hour and visualized under the Odyssey® Infrared Imaging System (Li-COR, Lincoln, NE).

Construction of mouse Tfeb promoter-driven reporter construct: Mouse genomic DNA isolated from primary mouse astrocytes was used as a template for PCR. 5 of the mouse TFEB (-916 / + 61) gene
'Adjacent sequences were isolated by PCR. Primers were designed from the sequence of the gene bank. Tfe
b: Sense: 5'-acgcgt CCA GGA GCC AGG GAC GGG GTA CAT CTC-3'(SEQ ID NO: 11);
Antisense: 5'-agatct AAG GAG AAA CTG AGT CCG GGC AGA AGG-3'(SEQ ID NO: 12)
.. Sense primers are tagged with Mlu1 restriction enzyme sites and antisense primers are BglII
Tagged with. PCR was performed using the Advantage-2 PCR kit (Clontech) according to the manufacturer's instructions. The resulting fragment was gel purified and ligated into a PGEM-TEasy vector (Promega). These fragments were further subcloned into the PGL-3 enhancer vector after digestion with the corresponding restriction enzymes and sequencing by sequencing (ACGT Inc. DNA Sequencinc Services).

Cloning of Tfeb promoter and site-specific mutagenesis: Site-specific mutagenesis was performed using a site-specific mutagenesis kit (Stratagene, USA). Two primers in opposite directions were used to amplify the mutated plasmid in a single PCR reaction. The primer sequences for the mutated promoter site are as follows: Mutant type: Sense: 5'-GCA AC
A GCA AGT GCG GAT TTG AGG GGG GGG GAC GGT GGG-3'(SEQ ID NO: 13); Antisense: 5'-CCC ACC GTC CCC CCC CCT CAA ATC CGC ACT TGC TGT TGC-3'(SEQ ID NO: 14). P
The CR product was precipitated with ethanol and then phosphorylated with T4 kinase. Phosphorylated fragments T4 D
The template was self-ligated with NA ligase and digested with the restriction enzyme DpnI to remove the unmutated template. The mutated plasmid was cloned and amplified in E. coli (DH5-α strain) competent cells.

Tfeb Promoter-Driven Reporter Activity Assay: Cells sown with 50-60% confluence in a 12-well plate using Lipofectamine Plus (Invitrogen) at 0.25 μg p.
TFEB (WT) -Luc and pTFEB (Mu) -Luc were co-transfected. 24 of transfection
After hours, cells were stimulated with different agents for 6 hours under serum-free conditions. Firefly luciferase activity in cell extracts was analyzed with TD-20 / 20 Luminometer (Turner Designs) using the Luciferase Assay System Kit (Promega) as described above (33, 34).

Chromatin immunoprecipitation assay: ChIP assay was performed using method (35) described by Nelson et al. With some modifications. Briefly, mouse primary astrocytes were stimulated with 10 μM gemfibrozil and 0.5 μM RA for 6 hours, then fixed with formaldehyde (1.42% of final volume) and quenched with 125 mM glycine. Pellet cells, 150 mM NaCl, 50 mM Tris-
Dissolved in IP buffer containing HCl (pH 7.5), 5 mM EDTA, NP-40 (0.5% vol / vol), Triton X-100 (1.0% vol / vol). 4.383 g NaCl, 25 ml 100 mM EDT for 500 ml
A (pH 8.0), 25 ml 1 M Tris-HCl (pH 7.5), 25 ml 10% (vol / vol) NP-40, and 50 ml 10% (vol / vol) containing the following inhibitors Triton X-100 was added: 10 μg / ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 30 mM p-nitrophenyl phosphate, 10 mM NaF, 0.1 mM Na ₃ VO ₄ , 0.1 mM Na ₂ MoO ₄ And 10 mM β-glycerophosphate.

After washing once with 1.0 ml IP buffer, 1 ml of pellets (containing all inhibitors) I
Resuspended in P-buffer and sonicated to divide the sheared chromatin into two fractions (
Use one as an input). Rotate the remaining fraction at 4 ° C. overnight at 5-7 μg of anti-PPARα or anti-RXRα antibody or anti-PGC1α or RNApol or normal IgG (Santa Cruz).
), Followed by rotation with Protein G-agarose (Santa Cruz) at 4 ° C. for 2 hours. The beads were then washed 5 times with cold IP buffer and
A total of 100 μl of 10% Chelex (10 g / 100 ml H ₂ O) was added directly to the washed protein G beads for vortexing. After boiling for 10 minutes, the Chelex / protein G bead suspension was cooled to room temperature. Proteinase K (100 μg / ml) was then added and the beads were incubated at 55 ° C. for 30 minutes with shaking and then boiled again for 10 minutes. The suspension was centrifuged and the supernatant was collected. The Chelex / protein G bead fraction was vortexed with another 100 μl of water and centrifuged again to combine the first and second supernatants. The eluate was used as is as a PCR template.

The following primers were used to amplify the flaking fragment of the RXR binding element in the mouse Tfeb promoter: Set 1: Sense: 5'-GAA CAT TCC AGG TGG AGG CA-3'
(SEQ ID NO: 15), antisense: 5'-CCC CCA ACA CAT GCT TCT CT-3'(SEQ ID NO: 16)
); Set 2: Sense: 5'-GAG TCT CTC GGA GGA GGT GA-3'(SEQ ID NO: 17), Antisense: 5'-ACT CCA GGC ATG CTT TCT CC-3'(SEQ ID NO: 18). PCR was repeated with varying numbers of cycles and different amounts of template to confirm that the results were within the linear range of PCR. qRT-P
CR was performed using the same primers and SYBR select master mix. The data were normalized for input and non-specific IgG and the rate of increase relative to the control was calculated.

Densitometry analysis: Protein blots were analyzed using ImageJ (NIH, Bethesda, MD) and bands were normalized to each β-actin loading control. The data show the mean magnification change relative to the control for at least 25 different images for each condition from 3 independent experimental sets.

Statistics: Values are shown as the mean ± SEM of at least 3 independent experiments. Statistical analysis of the difference was performed by Student's T-test. The standard for statistical significance was p <0.05.

[Example 2-Activation of PPARα and RXRα induces expression of TFEB in primary mouse brain cells]
PPAR activators such as the FDA-approved drug gemfibrozil were investigated to determine if TFEB expression in mouse brain cells could be upregulated. Since PPARα and RXRα are known to form transcriptionally active complexes (21, 36, 37), we use both gemfibrozil and ATRA to activate RXRα. It was checked whether the heavy treatment had any additive effect. Mouse primary astrocytes (MPA) were also treated in serum-free medium with single doses of gemfibrozil and ATRA, and also in combination. Quantitative real-time PCR data showed elevated expression of Tfeb in all three groups, with a slight increase in combination treatment (but not statistically significant for individual treatments) (Figure). 1A). When using a combination of both gemfibrozil and ATRA, we found that both compounds expressed similar levels of Tfeb at much lower doses (10 μM and 0.2 μM, respectively) compared to 25 μM gemfibrozil and 0.5 μM ATRA. I was able to achieve it. Time point analysis using the combined treatment showed that Tfeb expression could be induced from early 6 hours to 24 hours (Fig. 1D). QRT- for both dose and time
Evaluation of PCR data by Western blot showed a similar pattern of elevated TFEB levels (FIGS. 1B, 1C, 1E & 1F).

In addition, we used mouse primary astrocytes and primary neurons, which were treated with a combination of gemfibrozil and ATRA in serum-free medium for 24 hours to perform immunocytochemistry.
The data showed a clear increase in TFEB levels in both astrocytes and neurons, as well as the localization of TFEB in and around the nucleus (Fig. 1G & 1I). TFEB immunoreactivity was quantified using ImageJ, with an increase of about 4-fold in overall levels of TFEB and about 5-5-fold increase in TFEB localization in the treated nucleus.
A 6-fold increase was observed (Figs. 1H and 1J). It has been previously shown that starvation and nutrient deficiency lead to activation of TFEB, so in this study all untreated cells were similarly maintained under serum-free conditions during the treatment time and at TFEB levels. The changes in the baseline of were made to be the same between the groups.

[Example 3-PPARα and RXRα are involved in drug-mediated upregulation of TFEB]
The hypothesis that PPARα in combination with RXRα can be involved in drug-mediated upregulation of TFEB was used to knock down RXRα in WT mouse primary astrocytes using PPARα (-/-) animal-derived mouse primary astrocytes. Tested by doing. WT, PPARα (-/-) and PPARβ (-/-)
MPA obtained from animals was treated under the same conditions as above and checked for TFEB mRNA and protein expression. Both real-time PCR and Western blots for TFEB allowed TFEB to be upregulated in WT and PPARβ (-/-) astrocytes, but P
Not at the same level in PARα (-/-) astrocytes (Fig. 2A, 2B & 2C). This finding was further confirmed by immunocytochemistry, where WT and PPARβ (-/-) showed an almost 3- to 4-fold increase in TFEB levels but not PPARα (-/-) (Fig. 2F & 2G). ).

Since it has been reported that PPARγ-assisted activator-1a (PGC1A) can be involved in transcriptional activation of Tfeb, we determine whether PPARγ is involved in this particular drug-mediated TFEB expression.
Tested using a γ inhibitor. Western blots of TFEB when pretreated with a PPARγ-specific inhibitor prior to drug treatment suggest that gemfibrozil and ATRA may not utilize the PPARγ-mediated pathway for upregulation of TFEB. Shown (Figure 2D & 2E
). PPARα and RXRα are known to form transcriptional complexes, and our data are based on ATR.
It showed a slight increase in TFEB in the presence of A. We wanted to know if ATRA would work through RXRα. WT MPA was treated with RXRα-specific siRNA followed by a combination of gemfibrozil and ATRA, and both mRNA and protein analysis was performed. The data showed successful knockdown of the RXRα gene and, as a result, the effect of the drug was partially revoked in the absence of RXRα, as evidenced by Tfeb mRNA levels after RXRα silencing. (Fig. 2H & 2I). Western blots also showed similar results with significantly lower TFEB levels in RXRα silencing cells compared to after treatment with scrambled siRNA (Fig. 2J & 2K). Taken together, these data indicate that PPARα and RXRα may be involved in the upregulation of TFEB by gemfibrozil and ATRA.

[Example 4-PPARα / RXRα heterodimer transcriptionally regulates TFEB expression under treatment conditions]
Since PPARα and RXRα form a transcriptional complex together, we determined that these receptors would upregulate Tfeb and we tested whether this receptor transcriptionally regulates Tfeb expression. did. After analysis of the Tfeb promoter site, we found that the Tfeb transcription initiation site (TSS).
) Approximately 480 bp upstream from the peroxisomal growth factor response element (Peroxisomal Prolifera)
We found the existence of tor Response Element (PPRE). Tfeb promoter containing PERO (pTFEB (pTFEB (
WT))) was cloned into the pGL3 enhancer vector. We also mutated the core sequence of PPRE and cloned the mutated promoter construct (pTFEB (Mu)) into the PGL3 vector. The wild-type luciferase construct showed a marked increase in luciferase activity when transfected into BV2 cells (Fig. 3B). Cells containing the pTFEB (WT) luciferase construct were PPARα-antagonists (GW6471; 250nM), PPARβ-antagonists (GSK0660; 250nM).
), When treated with PPARγ-antagonist (GW9662; 5nM), luciferase activity similar to that of untreated cells was observed in cells treated with PPARα-antagonist, but PPARβ- or P
Not observed in cells treated with PARγ-antagonists (Fig. 3C).

In mouse primary astrocytes isolated from WT, PPARα (-/-) and PPARβ (-/-) animals, WT and
An increase in luciferase activity was observed in PPARβ (-/-) cells, but not in PPARα (-/-) (Fig. 3D). In addition, a construct with a mutant PPRE site (pTFEB (Mu) -Luc) was added to BV2.
And transfection into mouse primary astrocytes, a dramatic reduction in luciferase activity was found in cells containing mutant constructs (Fig. 3E & 3F). In summary, from these data,
It is shown that activation of PPARα plays an important role in the induction of Tfeb after treatment with gemfibrozil and retinoic acid.

Finally, we decided to investigate the role of the actual DNA binding of PPARα to the Tfeb promoter in this situation. After activation of PPARα, RXRα and PGC1α have been shown to form a complex that initiates transcriptional activation of many genes (38-42); we examine whether this is also the case here. did. Mouse primary stellate cells treated with gemfibrozil and retinoic acid at various time points from 30 minutes to 240 minutes were subjected to ChIP analysis by immunoprecipitating chromatin fragments with anti-PPARα, -RXRα, and -PGC1α antibodies. -RNA Pol and normal IgG were used as controls. Both semi-quantitative PCR and quantitative RT-PCR showed that pull-down with specific antibodies increased amplicon enrichment over time (Fig. 4B & 4C). Normal Ig next to immunoprecipitation
PCR using G showed a band that was almost undetectable by RT-PCR, and P using fully fragmented DNA.
In CR, RT-PCR showed a homogeneous signal, showing the uniformity and specificity of the results. In real-time PCR, the Ct value was normalized to% with respect to the input, and further normalized using an IgG signal to obtain a signal exceeding the noise value, demonstrating the specificity of the result. The experiment was repeated at least 3 times under the same conditions and cycles, and the dilution of the PCR product was adjusted so that the data were within the linear range of PCR. All previous findings indicate that activation of PPARα and RXRα receptors can actually directly regulate Tfeb expression transcriptionally.

[Example 5-TFEB upregulation leads to increased lysosomal biosynthesis]
Because TFEB is the main regulator of lysosomal gene expression and biosynthesis (9, 12)
, 16), we predicted an increase in lysosomal marker biosynthesis with upregulation of TFEB. MPA treated under the same conditions was subjected to several lysosomal markers, such as Lamp2.
, Limp2 and Npc1 were analyzed for mRNA. As expected, from the data, WT and PPARβ (-/
-) Elevated levels of these genes were shown under treatment conditions in cells, but not in PPARα (-/-) cells (Fig. 5A). Western blot analysis and immunocytochemistry for LAMP2 in WT and KO cells also showed similar protein expression patterns (Fig. 5B, 5C & 5D).
). Elevated levels of LAMP2 were also observed in primary mouse neurons (Fig. 5E). Furthermore, when cells were stained with lysotracker red, an increase in lysosomal content per cell was observed for drug-treated WT and PPARβ (-/-) cells, but not for PPARα (-/-). (Fig. 5F
), This is consistent with our previous findings on TFEB and other lysosomal markers. These data suggest that gemfibrozil and ATRA can induce TFEB expression via the PPARα / RXRα pathway, which ultimately leads to increased lysosomal biosynthesis.

[Example 6-PPARα and RXRα agonists are in wild-type and PPARβ / -mouse CNS.
Induces lysosomal biosynthesis in vivo, but not in PPARα-/-mice]
Since the involvement of PPARα was confirmed in the upregulation of fibrate-mediated TFEB, we further checked whether the same results could be reproduced in vivo situations. Also used as a vehicle in WT, PPARα (-/-) and PPARβ (-/-) mice of the same background 0.
7.5 mg / kg bw / day of gemfibrozil dissolved in 1% methylcellulose and 0.1 mg / kg bw
ATRA was orally administered for 60 days. At the end of the procedure, the mice were sacrificed, the cerebral cortex was sectioned, and TFEB
Immunofluorescence was performed for the presence of. No significant increase in TFEB levels was observed in the cortex of PPARα (-/-) treated animals compared to vehicle controls, but a significant response was observed in WT and PPARβ (-/-) animals (Fig. 6A, Figure 6A, 6D & 6G), this in vivo immunohistochemical data confirmed our findings in cell culture.

We further quantified TFEB-positive signals in at least 12 sections per group and expressed the numbers as a percentage of total area. Quantitative analysis confirmed a significant increase in TFEB-positive signals in WT and PPARβ (-/-) animals, but not in PPARα (-/-) animals (Fig. 6B, 6E & 6H). Other sections of the cortex of the same animal were subjected to immunohistochemistry for the presence of LAMP2. The results show an increase in LAMP2 immune reactivity in WT and PPARβ (-/-) animals, but not in PPARα (-/-) animals (Fig. 7A, 7D & 7G). From quantitative data, WT
It was suggested that the LAMP2-positive signal was significantly increased in PPARβ (-/-) and not in PPARα (-/-) animals (Fig. 7B, 7E & 7H).

[Example 7-Gemfibrozil and ATRA induced lysosomal biosynthesis in fibroblasts of LINCL patients]
To test whether similar results can be reproduced in patient cells, we are normal and LINCL
Obtained skin fibroblasts from affected patients and reduced serum media (2% serum)
Cells were treated with similar concentrations of gemfibrozil and ATRA. Serum deficiency (starvation)
), Untreated cells were maintained under similar serum conditions during the treatment time (24 hours). Fibroblasts were then stained with lysotracker red and a similar pattern of increased intracellular lysosomal accumulation was observed throughout the board. Normal fibroblasts (WT # 1)
~ WT # 3) and LINCL patient-derived fibroblasts (NCL # 1 to NCL # 5) with Cln2 mutations and LINCL
Carrier (NCL / C) fibroblasts showed a similar increase in lysosomes per cell (Fig. 8). To normalize the number and size of cells in the image, we calculated the lithotracker positive signal per unit area per cell and analyzed the magnification relative to the control (fold).
over control analysis). Analysis of lysosomal positive signals in at least 25 visual fields per group and data suggested a significant increase in all fibroblasts regardless of disease status, but basal levels of intracellular lysosomes and elevations. Levels were different from cell to cell. This data suggests that the effect of the treatment is independent of the disease state of LINCL patients.

[Discussion of Example 8-Examples 2 to 7]
Lysosomes not only function as a cell waste treatment mechanism, but also include antigen presentation, regulation of specific hormones, bone remodeling, necrosis cell death, cell surface repair, and development and other signaling pathways. It is one of the major intracellular organs that also plays an important role in the biological process of lysosomes (2, 43-47). Lysosome biosynthesis and activity need to be tightly regulated to perform these various functions. Recent findings indicate that TFEB is a major regulator of lysosomal biosynthesis (9, 12, 15). Over the years, different groups have emphasized the role of lysosomes in different disease situations (48-53). Lysosome-related genes have been reported to be closely regulated in the orbital fat pad of patients with Graves' ophthalmopathy, while downregulation of processing in lysosomes
Improved transfection of PEGylated lipopolyplex-mediated genes (53, 54). Increased lysosomal biosynthesis may not necessarily prove beneficial in all diseases and cell types, but in some cases the induction of the autophagy-lysosomal pathway is due to clearance of toxic waste from cells. Could be useful (55, 56).

Over the past few years, TFEB has emerged as a potential therapeutic target for several lysosome-related diseases. Taiji Tsunemi et al. Reported that activation of the transcription factor EB (TFEB) via PGC1α could result in increased htt turnover and elimination of protein aggregates (57, 58). There are reports suggesting a link between α-synuclein toxicity and TFEB dysfunction, identifying TFEB as a target for neuroprotective therapy in PD (59). TF
Activation of EB has been shown to promote folding, trafficking and activity of destabilizing glucocerebrosidase (GC) variants in Gaucher's disease. Another LS
In the case of Tay-Sachs disease, which is D, TFEB has been shown to restore the activity of β-hexosaminidase mutants. These findings describe TFEB as a specific regulator of lysosomal protein homeostasis and as a therapeutic target for restoring enzymatic homeostasis in LSD (60, 61).

It has also been reported that induction of lysosomal exocytosis by overexpression of TFEB can restore pathological storage in LSD and restore normal cell morphology (62).
). Apart from LSD, TFEB has been shown to induce lipid catabolism and clearance.
It has been able to treat obesity and metabolic syndrome in mice (15, 16). Overall, in recent years, due to its role, TFEB has the potential to be an important transcription factor not only for lysosomal biosynthesis, but also as a therapeutic target in disease states due to its association. ing. Although many therapeutically usable compounds targeting TFEB activity have not been identified,
A recently approved excipient by the FDA, 2-hydroxypropyl-β-cyclodextrin (HP)
βCD) has been shown to promote TFEB-mediated clearance of proteolipid aggregates in cells of patients with LINCL (56). In another study, mucopolysaccharidosis (
By genistein (5,7-dihydroxy-3- (4-hydroxyphenyl) -4H-1-benzopyran-4-one), a potential drug for use in substrate reduction therapy (SRT) for MPS)
It was revealed that TFEB levels and activity, as well as lysosomal biosynthesis, were induced (63).
..

Recent studies have linked TFEB, lysosomal biosynthesis and autophagy to lipid metabolism (14-16, 55, 64, 65). Due to the potential interaction of TFEB with lipid metabolism, we
We decided to investigate the roles of gemfibrozil and ATRA, which are potential activators of PPARα and RXRα, two important factors in lipid metabolism. Gemfibrozil, marketed as "Lopid," is an FDA-approved drug prescribed for dyslipidemia (17, 19).
It has been shown to have multiple beneficial effects (22). Gemfibrozil is the blood-brain barrier (
The ability to pass BBB) has already been reported (20). We have previously reported that gemfibrozil in combination with ATRA can induce levels of the Cln2 gene in brain cells (66).
). We further investigated whether TFEB, the major regulator of lysosomal biosynthesis, could be affected by the drug. From our data, Gemfibrozil,
It is shown that the expression of TFEB in brain cells could be effectively induced alone or in combination with ATRA.

Further studies suggest a potential role for PPARα in this process. PPAR
α has been shown to play an important role in various regulatory pathways (67-71).
). Certain polyunsaturated fatty acids and oxidized derivatives, and fibrate family lipid modifiers, including fenofibrate and gemfibrozil, are known to activate PPARα. Fibrates replace the HSP90 repressor complex that sequesters PPARα in the cytosol and helps restore the transcriptional activity of PPARα (21). Therefore, we evaluated the role of the PPAR group of receptors in this phenomenon. From our data, PPARα is involved in the process of upregulation of TFEB by gemfibrozil, but PPARβ and PPARγ.
Is clearly shown not to be involved. This in vitro study was further validated by in vivo studies using PPARα and PPARβ knockout mice. Our in vivo results also supported the cell culture data.

To explain the mechanism of TFEB upregulation, the promoter region of the Tfeb gene was analyzed. A PERO site and an RXR binding site were found in the mouse Tfeb promoter. Previous studies showed that PPAR / RXR heterodimers showed DNA-binding activity (70). PPAR / RXR heterodimers also regulate the transcription of genes whose products are involved in lipid homeostasis, cell proliferation, and differentiation (69,72). The route of Tfeb upregulation is PPAR
And RXR were observed to require a joint action. Moreover, the effects of both gemfibrozil and RA were revoked in the absence of either RXRα or PPARα. Results of a luciferase assay using PERO's WT on the Tfeb promoter and a mutant luciferase construct showed an increase in Tfeb promoter-dependent activity after stimulation with the WT construct. However, PPARα (-/-) cells transfected with the pTFEB (WT) -Luc construct, and pTFEB (
WT cells transfected with the Mu) -Luc construct did not show a significant increase in luciferase activity. Finally, from the ChIP data, TF of PPARα and RXRα along with PGC1α and RNA Pol
Recruitment of the EB promoter onto the PERO site was shown. Experimental data were rigorously analyzed incorporating appropriate controls to ensure the specificity of the findings.

Taken together, these data are based on the PPARα activator gemfibrozil and RXRα.
ATRA, an agonist of ATRA, describes a unique mechanism that together upregulates TFEB in brain cells via PPARα / RXRα heterodimers. One study reported that PPARγ-null trohoblast stem (TS) cells had low levels of TFEB on day 4 of differentiation, but a study using GW9662, a potent known PPARγ antagonist in brain cells, produced PP.
Substantial involvement of ARγ was not revealed (73). This may be due to differences in cell type, ie, differences between differentiated TS cells and mature primary astrocytes / neurons, or differences in the level of potential for activation of PPARα. According to a comprehensive study by Settembre et al., The authors found that PPARα and PGC1α are targets of TFEB under starvation-induced stress, and that TFEB is self-regulating in the case of starvation stress. As reported, another study by Tsunemi et al. Placed PGC1α upstream of TFEB in the context of Huntington's disease. It is quite possible that TFEB regulates lipid metabolism via PPARα and PGC1α, both of which have very important roles in the regulation of lipid metabolism. However,
On the other hand, the data in this application show that direct stimulation of PPARα induces recruitment of the PPARα-RXRα-PGC1α complex onto the TFEB promoter, thereby affecting lysosomal biosynthesis.

Although the stress response directly regulates TFEB function, the findings also indicate that externally stimulated activation of PPARα and RXRα can also regulate TFEB, which in turn regulates the expression of PPARα or other genes involved in lipid metabolism. Suggest to get. However, more detailed studies are needed to interpret the existence of such feedforward control mechanisms and the apparent bidirectional interaction of lipid metabolism with lysosomal biosynthesis.

In summary, this study tested a novel hypothesis that lipid-lowering agents such as gemfibrozil can upregulate lysosomal biosynthesis in brain cells through PPARα-mediated activation of TFEB. Given the important role that TFEB plays in a particular disease situation,
There is growing interest in identifying TFEB as a therapeutic target. The results of this study reveal unknown properties of PPARα, describe new therapeutic options for LSD, reveal more dramatic regulation of TFEB, and interest in understanding the relationship between lipid metabolic pathways and lysosomal biosynthesis. To increase.

[Example 9-Upregulation of TFEB mRNA expression in mouse astrocytes by a cholesterol-lowering agent]
FIG. 9 shows cholesterol lowering agents (simvastatin and pravastatin), aspirin (
TFEB in mouse stellate cells with analgesics and antipyretics), cinnamic acid (cinnamon metabolite), and drugs for urea cycle disorders (sodium phenylbutyrate and sodium benzoate)
Shows upregulation of mRNA expression. Mouse primary stellate cells with various concentrations (see Figure 9) of simvastatin and pravastatin (A), (B), cinnamic acid (C), and sodium phenylbutyrate and sodium benzoate (D) under serum-free conditions. After incubating for 5 hours
TFEB mRNA expression was monitored by semi-quantitative RT-PCR. Sodium formate (D) was used as a negative control for sodium phenylbutyrate and sodium benzoate.

[Example 10-Aspirin induces lysosomal biosynthesis in primary mouse astrocytes]
We investigated whether aspirin, one of the most widely used drugs in the world, could upregulate lysosomal biosynthesis in mouse brain cells. Astrocytes were treated with various doses of aspirin in serum-free medium and then stained with lyso-tracker. As evidenced by increased lysosomal staining, aspirin markedly increased lysosomal biosynthesis in astrocytes at doses of 2 and 5 μM (Fig. 10). However, 10 μM
At this dose, aspirin was not very potent in increasing lysosomal biosynthesis (figure).
Ten).

[Example 11-Aspirin increases LAMP2 expression in primary mouse astrocytes]
LAMP2 is an important lysosomal membrane protein that plays a key role in the formation of new lysosomes. We observed an increase in LAMP2 mRNA (Fig. 11A) and protein (Fig. 11C) expression by aspirin over time in astrocytes. From dose-dependent studies, 2 and 5
Aspirin at a dose of μM showed increased LAMP2 protein expression (Fig. 11B). The increase in LAMP2 by aspirin was further confirmed by immunostaining (Fig. 11D).

[Example 12-Aspirin upregulates TPP1 expression and activity in primary mouse astrocytes]
Tripeptidyl peptidase 1 (TPP1) is a target molecule in this disease because the activity of this protein is deficient in late-onset childhood Batten disease. We investigated whether aspirin could upregulate TPP1 in astrocytes.
Dose-dependent studies showed an increase in TPP1 protein at low doses of aspirin (2 and 5 μM) (Fig. 12A). The increase in TPP1 protein in response to 5 μM aspirin was apparent as early as 2 hours and was maximal after 12 hours of incubation (Fig. 12B). Again, we are 2 and
An increase in TPP1 activity was observed at 5 μM aspirin, but not at 10 μM (Fig. 12C).
..

[Example 13-Aspirin upregulates TFEB expression in primary mouse astrocytes]
Recent findings indicate that TFEB is a major regulator of lysosomal biosynthesis (9, 12).
, 15). Over the years, different groups have emphasized the role of lysosomes in different disease situations (48, 49, 52, 53). Lysosome-related genes have been reported to be closely regulated in the orbital fat pad of patients with Graves' ophthalmopathy, while PEGylated lipopolyplex-mediated genes due to downregulation of processing in lysosomes. Transfection is improved (53, 54). Increased lysosomal biosynthesis may not necessarily prove beneficial in all diseases and cell types, but in some cases the induction of the autophagy-lysosomal pathway is due to clearance of toxic waste from cells. Could be useful (55, 56). Over the past few years, TFEB has emerged as a potential therapeutic target for several lysosome-related diseases. According to Tsunemi et al. (57), activation of the transcription factor EB (TFEB) via PGC1α can result in increased htt turnover and elimination of protein aggregates. There is a report that suggests a relationship between α-synuclein toxicity and TFEB dysfunction and identifies TFEB as a target for neuroprotective therapy in PD (
59). Activation of TFEB has also been shown to promote folding, trafficking and activity of destabilizing glucocerebrosidase (GC) variants in Gaucher's disease. In the case of another LSD, Tay-Sachs disease, TFEB has been shown to restore the activity of β-hexosaminidase mutants. These findings describe TFEB as a specific regulator of lysosomal protein homeostasis and as a therapeutic target for restoring enzymatic homeostasis in LSD (60, 61).

Therefore, we investigated whether aspirin could upregulate TFEB in astrocytes. Dose-dependent studies have shown that aspirin can increase TFEB protein levels at doses of 2 and 5 μM (Fig. 13A). Immunofluorescence analysis further confirmed the upregulation of TFEB by aspirin (Fig. 13B). We also cloned the Tfeb promoter into a pGL3 enhancer vector and investigated the reporter activity driven by the Tfeb promoter. Interestingly, aspirin induces Tfeb promoter-driven luciferase activity in astrocytes (Fig. 13C), suggesting that aspirin increases transcription of the Tfeb gene.

[Example 14-Aspirin induces activation of PPARα in primary mouse astrocytes]
In recent years, we have discovered that PPARα plays an important role in the transcription of Tfeb. Therefore, we here examined whether aspirin can induce PPARα activation in astrocytes. First, we used an electrophoretic mobility shift assay (EMSA) to monitor the DNA-binding activity of PPARα and found an increase in PPARα activation by aspirin over time (Fig. 14A). To confirm these results, we have WT, PPARα (-/-) and PPARβ.
(-/-) Astrocytes were isolated from mice and the transcriptional activity of PPAR was monitored. Interestingly, aspirin increased PPRE-driven luciferase activity in astrocytes isolated from WT and PPARβ (-/-) mice, but not in PPARα (-/-) mice (Fig. 14B). ), It was suggested that aspirin can induce the activation of PPARα, but not the activation of PPARβ.

[Example 15-Aspirin upregulates TFEB in primary mouse astrocytes via PPARα]
Astrocytes isolated from WT, PPARα (-/-) and PPARβ (-/-) mice were treated with 5 μM aspirin followed by immunofluorescence analysis of TFEB. In Figure 15, aspirin is WT and PPARβ (-/-
) Induced the expression of TFEB in astrocytes isolated from mice, but not in PPARα (-/-) mice. These results indicate that aspirin requires PPARα to increase TFEB in astrocytes, but not PPARβ.

[Example 16-Aspirin increases lysosomal biosynthesis in primary mouse astrocytes via PPARα]
Because TFEB is the main regulator of lysosomal gene expression and biosynthesis (9, 12,
16), we investigated the effect of aspirin on lysosomal biosynthesis. WT, PPARα (-/-)
And astrocytes isolated from PPARβ (-/-) mice were treated with different doses of aspirin and then LAMP2 levels were monitored. As is clear from Figures 16A-B, aspirin is WT and
The level of LAMP2 in astrocytes isolated from PPARβ (-/-) mice was increased in a dose-dependent manner, but not in PPARα (-/-) mice. We also examined the status of lysosomes by lysosomal staining. Similar to the LAMP2 results, aspirin is WT and PPARβ (
-/-) Increased lysosomal biosynthesis in astrocytes isolated from mice, but with PPARα (-
/-) Not increased in mice (Fig. 17).

[Discussion of Example 17-Examples 10 to 16]
Aspirin has several advantages compared to other available therapies for lysosomal storage diseases. On the other hand, aspirin has been widely used as an analgesic for decades around the world.
On the other hand, aspirin can be taken orally, which is the least painful route. Aspirin has been reported to exhibit some toxic effects at high doses (gastric ulcer, gastric bleeding, tinnitus, etc.) (74), but in our study, aspirin at low doses (2 and 5 μM).
) Promotes lysosomal biosynthesis, and aspirin may not be toxic at low doses. However, even at low doses of aspirin, some toxic effects (eg gastric ulcer)
If, there is a way to reduce the side effects. For example, for oral use
And to avoid degradation in the stomach, enteric coated aspirin is available. In randomized open trials, low doses of aspirin in sustained release formulations have been shown to be effective as antiplatelet agents with no significant side effects (75). One study (76) used S-adenosyl-methionine (SAM), an amino acid that is naturally formed in the body, and was given a dose of 500 mg SAM with a high dose of aspirin (1300 mg) to cause stomach damage. Has been found to be reduced by 90%.

本発明をその具体的な例示的実施形態を参照して記載及び説明したが、本発明がこれら
の例示的実施形態に限定されることを意図するものではない。当業者であれば、添付する
請求の範囲によって規定される本発明の範囲及び精神から逸脱することなく変更及び改変
が可能であることを認識するであろう。従って、そのような変更及び改変の全てが添付す
る請求の範囲及びその等価物の範囲内に含まれるように本発明に含まれることが意図され
る。 In summary, we found that the widely used analgesic aspirin is a PPARα-mediated TF.
It was demonstrated to increase lysosomal biosynthesis in astrocytes through upregulation of EB. These results emphasize that this drug can be used as the primary or adjunctive treatment for therapeutic intervention in late-onset childhood Batten disease and other LSDs.

Although the present invention has been described and described with reference to its specific exemplary embodiments, it is not intended that the invention be limited to these exemplary embodiments. Those skilled in the art will recognize that modifications and modifications can be made without departing from the scope and spirit of the invention as defined by the appended claims. It is therefore intended to be included in the invention such that all such modifications and modifications are within the scope of the appended claims and their equivalents.

Claims (26)

A method for the treatment of lysosomal storage diseases, in which a therapeutically effective amount of a composition containing an agent that mediates upregulation of the transcription factor EB is administered to a subject in need of such treatment. Including, the above method. The method of claim 1, wherein the agent is a statin. The method of claim 2, wherein the statin is selected from the group consisting of atrovastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simuvastatin, and combinations thereof. The method of claim 1, wherein the agent is selected from the group consisting of analgesics, antipyretics, aspirin, cinnamon metabolites, cinnamic acid, sodium phenylbutyrate and sodium benzoate. The method of claim 1, wherein the agent is a lipid lowering agent. The method of claim 5, wherein the lipid lowering agent is a fibrate. The method of claim 6, wherein the fibrate is gemfibrozil or fenofibrate. The method according to any one of claims 1 to 7, wherein the composition further contains a therapeutically effective amount of all-trans retinoic acid or vitamin A. The composition contains fibrates and all-trans retinoic acid or vitamin A.
The method according to claim 8. 9. The method of claim 9, wherein the composition comprises a synergistic combination of fibrates and all-trans retinoic acid or vitamin A. Lysosome storage disease includes Parkinson's disease including neuroseloid lipofustinosis, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis (ALS), multiple system atrophy (MSA), and progressive supranuclear disease. The method according to any one of claims 1 to 10, which is a neurodegenerative disease selected from the group consisting of paralysis (PSP), corticobasal degeneration (CBD) and Lewy body dementias (DLB). The method of any one of claims 1-10, wherein lysosomal storage disease is a disorder of the autophagy pathway and the agent increases lysosomal biosynthesis. Lysosomal storage diseases include Tay-Sachs disease, Fabry disease, Niemann-Pick disease, Gaucher disease, Hunter syndrome, α-mannose disease, and Aspartylgluc
osaminuria), Cholesteryl ester storage disease
The method according to any one of claims 1 to 10, selected from the group consisting of chronic hexosaminidase A deficiency, cystinosis, Danon disease, Farber's disease, fucosidosis, and galactosialidosis. Transcription factor EB increases transcription factor EB mRNA levels, transcription factor EB protein levels,
Alternatively, claims 1-1, which are up-regulated by activation of the PPARa-RXRa heterodimer.
The method according to any one of 0. A method for the treatment of lysosomal storage diseases, which comprises administering a composition containing a therapeutically effective amount of the drug to a subject in need of such treatment, wherein the drug restores transcription factor EB activity. The above method. 15. The method of claim 15, wherein the agent is fibrate. 16. The method of claim 16, wherein the fibrate is gemfibrozil or fenofibrate. 16. The method of claim 16, wherein the therapeutically effective amount of fibrate is lower when the fibrate is administered in combination with all-trans retinoic acid or vitamin A. A method for the treatment of lysosomal storage diseases, which comprises administering to a subject in need of such treatment a composition containing a therapeutically effective amount of a drug that mediates gene upregulation. , The method described above, wherein the gene is the Tfeb gene. Lysosome storage disease includes Parkinson's disease including neuroseloid lipofustinosis, Alzheimer's disease, Huntington's disease, muscular atrophic lateral sclerosis (ALS), multiple system atrophy (MSA), and progressive supranuclear disease. 19. The method of claim 19, wherein the neurodegenerative disease is selected from the group consisting of paralysis (PSP), cerebral cortical basal nucleus degeneration (CBD) and Lewy body dementias (DLB). A combination of drugs containing statins and all-trans retinoic acid or vitamin A. The combination of pharmaceuticals according to claim 21, further comprising a pharmaceutically acceptable carrier. The combination of medicines according to claim 21, wherein the combination of medicines is a synergistic combination of medicines. The method of claim 4, wherein the agent is aspirin. Lysosomal storage diseases include Tay-Sax disease, Fabry disease, Niemann-Pick disease, Gaucher's disease, Hunter syndrome, α-mannose disease, aspartyl glucosamineuria, cholesterol ester storage disease, chronic hexosaminidase A deficiency, cystinosis. , Danon disease, Farber's disease, fucosidosis, galactosialidosis, and any one of claims 1-10 selected from the group consisting of Batten disease including late-onset childhood Batten disease and juvenile Batten disease
Item description method. Lysosomal storage diseases include Tay-Sachs disease, Fabry disease, Niemann-Pick disease, Gaucher's disease, Hunter syndrome, α-mannose disease, aspartyl glucosamineuria, cholesterol ester storage disease, chronic hexosaminidase A deficiency, cystinosis. 15. Method.

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